Effects of adenovirus-mediated p16INK4A expression on cell cycle arrest are determined by endogenous p16 and Rb status in human cancer cells.
We constructed an adenoviral vector containing human p16 cDNA in order to evaluate the cytotoxic effects of exogenous p16 expression on cancer cell proliferation and to explore the potential use of p16 in cancer gene therapy. Following infection of human breast (MCF-7, MDA-MB-231, and BT549), osteosarcoma (U-2 OS and Saos-2), cervical (C33a), and lung cancer (H358) cell lines with the recombinant adenovirus Adp16, high levels of p16 expression were observed in all cell lines. Cancer cell lines which were mutant or null for p16 but wild-type for the retinoblastoma gene product (pRb) (MCF-7, MDA-MB-231, BT549 and U-2 OS) were 7-22-fold more sensitive to the cytotoxic effects of Adp16 than to a control virus. In contrast, cancer cell lines which were wild-type for p16 but mutant or null for pRb (Saos-2, C33a and H358) were <threefold more sensitive to Adp16 when compared to a control virus. Analysis of 5-bromodeoxyuridine incorporation into DNA following infection with Adp16 showed a loss of S phase in those cell lines which were null or mutant for p16 but expressed a functional pRb. This cell cycle arrest was associated with binding of the p16 protein to cyclin-dependent kinase 4 and dephosphorylation of pRb. In contrast, human cancer cell lines expressing a wild-type p16 and a mutant pRb or no pRb showed no substantial loss of S phase following Adp16 infection. Based on these studies, we conclude that p16-mediated cytotoxicity is tightly associated with the presence of functional pRb in human cancer cells, and that tumor cells which are mutant or null for p16 are candidates for Adp16 mediated cancer gene therapy.
Craig
C
C
Medicine Branch, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892, USA.
Kim
M
M
Ohri
E
E
Wersto
R
R
Katayose
D
D
Li
Z
Z
Choi
Y H
YH
Mudahar
B
B
Srivastava
S
S
Seth
P
P
Cowan
K
K
D000256:Adenoviridae / Q000235:genetics*; D001973:Bromodeoxyuridine / Q000378:metabolism; D002453:Cell Cycle; D051358:Cyclin-Dependent Kinase 4; D019941:Cyclin-Dependent Kinase Inhibitor p16 / Q000032:analysis / Q000235:genetics*; D018844:Cyclin-Dependent Kinases / Q000378:metabolism; D015316:Genetic Therapy; D006801:Humans; D009369:Neoplasms / Q000628:therapy*; D010766:Phosphorylation; D011518:Proto-Oncogene Proteins; D016160:Retinoblastoma Protein / Q000032:analysis*; D014407:Tumor Cells, Cultured; D016159:Tumor Suppressor Protein p53 / Q000032:analysis
D019941:Cyclin-Dependent Kinase Inhibitor p16; D011518:Proto-Oncogene Proteins; D016160:Retinoblastoma Protein; D016159:Tumor Suppressor Protein p53; C495900:CDK4 protein, human; D051358:Cyclin-Dependent Kinase 4; D018844:Cyclin-Dependent Kinases; D001973:Bromodeoxyuridine